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目的:测定抗Ⅳ型胶原酶单链抗体Fv和力达霉素辅基蛋白LDP在大肠杆菌中表达产生的融合蛋白Fv-LDP含量。方法:3瓶培养融合蛋白Fv-LDP表达菌,固定化金属亲和层析纯化融合蛋白Fv-LDP,分步透析使之复性,HPLC测其纯度。比色法测定菌体浓度,SDS-PAGE分离蛋白带,积分密度扫描法测定融合蛋白Fv-LDP表达百分比和含量。结果:在88.7~1 064 mg.L-1范围内,融合蛋白Fv-LDP浓度和积分密度值呈线性关系(r=0.998 8),精密度RSD为2.09%(n=8),平均回收率为95.18%。结论:SDS-PAGE-SpotDenso法操作简单,重复性好,灵敏度高,适用于发酵过程中对融合蛋白Fv-LDP表达产量的控制分析。
OBJECTIVE: To determine the content of Fv-LDP fusion protein produced by the expression of anti-type IV collagenase Fv and levomycin-dependent protein LDP in E.coli. Methods: The fusion protein Fv-LDP was expressed in 3 bottles and the fusion protein Fv-LDP was purified by immobilized metal affinity chromatography. The purity was determined by HPLC. The concentration of Fv-LDP and the content of Fv-LDP fusion protein were determined by colorimetric method. Results: In the range of 88.7-1,064 mg · L-1, the concentration of Fv-LDP showed a linear relationship with the integral density (r = 0.998 8) and the RSD of precision was 2.09% (n = 8) 95.18%. Conclusion: SDS-PAGE-SpotDenso method is simple, reproducible and sensitive. It is suitable for the control analysis of Fv-LDP expression in the fermentation process.